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H. Yang
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P3.03 - Poster Session with Presenters Present (ID 473)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.03-008 - Hypoxia-Induced Changes in microRNA Levels Contribute to Drug Resistance in a 3D Model of Malignant Pleural Mesothelioma (ID 5867)
14:30 - 14:30 | Author(s): H. Yang
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is an aggressive asbestos-related thoracic cancer. Chemotherapy is the most frequent treatment option but almost every patient will be confronted with recurrence of disease and drug resistance. Previous studies have used 3D spheroid cultures to investigate drug response in MPM. We showed that microRNAs are important players in MPM biology and that they contribute to the response of MPM cells to some chemotherapy drugs. In the current study we aimed to investigate the role of microRNAs in the drug resistance of a 3D spheroid model of MPM.
Methods:
MPM cells were grown in standard 2D culture or as 3D spheroids in low adherence round bottom multi-well plates. The structure of the spheroids was confirmed by conventional and scanning electron microscopy. MicroRNA expression was profiled using TaqMan Low Density Arrays. RT-qPCR and droplet digital PCR were used to validate candidate microRNAs. HIF1a expression was examined in MPM spheroids using immunofluorescence staining. Drug cytotoxicity was investigated in both 2D and 3D cultures using standard proliferation assays, and the effect of drugs on gene expression was analysed. MicroRNA mimics and siRNAs were used to determine the influence of microRNA and HIF1a expression on drug resistance.
Results:
In our adapted model of 3D cell growth, MPM cell lines formed spherical 3D structures, in contrast to the donut shapes reported with other models. MPM cells in these spheroids were more resistant to cisplatin and gemcitabine when compared to cells grown in 2D cultures. Immunofluorescence revealed a hypoxic gradient with high HIF1a expression observed in the centre of the spheroids. Spheroids also exhibited a significant up-regulation of miR-210, miR-21, miR-378a, miR-195 and miR-146b, and down-regulation of miR-320b and miR-1225b. Transfecting MPM cells in 2D culture with miR-210 or miR-21 mimics resulted in increased drug resistance, whereas HIF1a knockdown inhibited spheroid formation and decreased drug resistance. Spheroids displayed higher expression of the ABCG2 drug pump, and ABCG2 was also up-regulated in cisplatin and gemcitabine treated MPM cells.
Conclusion:
Our spheroid model revealed a clear impact of hypoxia on gene expression in MPM cells. Hif1a was highly expressed in the hypoxic centre of the spheroids and is an upstream regulator of the microRNAs we found to be differentially expressed. Pharmacologic and genetic modulation of microRNA and HIF1a levels altered drug resistance in MPM cells, suggesting a link between hypoxia, microRNAs and drug resistance in MPM.
